Summary/Abstract Human chromosomes end in telomeres, repetitive DNA sequences that are bound by the Shelterin protein complex (1). During semi-conservative DNA replication the extreme ends of a chromosome are unable to be duplicated, leading to successive chromosome shortening. Once telomeres reach a critical length, cells enter senescence or undergo apoptosis (2). To counteract chromosome shortening, continuously dividing cells, such as germ cells, stem cells, and most cancer cells, express telomerase, an RNA-containing reverse transcriptase (3). Telomerase is a unique enzyme that processively adds telomeric repeats, copied from its RNA component, to the single-stranded DNA overhang of chromosome ends (4). The molecular mechanisms that govern telomerase processivity are poorly defined, but are critical to understand telomere maintenance. The Shelterin complex carries out two key functions at telomeres; it prevents telomeres from being recognized as sites of DNA damage, and it recruits telomerase to telomeres (5,6). Telomerase recruitment to telomeres is a tightly regulated process. Telomerase resides in Cajal bodies, specialized RNA-processing compartments in the nucleus, throughout most of the cell cycle. During S-phase, telomerase is recruited to telomeres to maintain telomere length (7). Although the protein-protein interactions required for telomerase to associate with telomeres are well understood, the spatio- temporal control of telomerase recruitment is poorly defined (7). Potential mechanisms for regulating telomerase recruitment include alterations in composition of telomerase and the shelterin complex or post-translational modification of its components. Telomere maintenance plays an important role in multiple human diseases. Deficiencies in telomerase assembly, activity, or recruitment to telomeres cause dyskeratosis congenita, pulmonary fibrosis, and aplastic anemia, severe human conditions characterized by stem cell failure (8). In addition, 90% of cancers rely on telomerase activity to allow them to divide indefinitely (9). Therefore, understanding the basic biology of telomerase recruitment to telomeres and telomerase catalysis could lead to novel approaches to modulate this process as a therapeutic approach for several human diseases. I propose to analyze the molecular mechanisms underlying telomerase recruitment to telomeres and telomerase catalysis using genome editing and a combination of cell biological, proteomic, biochemical, and single-molecule approaches. In particular I will: 1. Determine the molecular mechanisms that drive telomerase recruitment to telomeres in S- Phase. Using genome-edited cell lines expressing tagged telomerase and shelterin components, I will conduct live cell imaging of telomerase trafficking to telomeres, analyze the assembly state of telomerase and the shelterin complex throughout the cell cycle using cell biological and proteomic approaches, and identify kinases that modulate telomerase trafficking. 2. Define the biochemical and biophysical properties of telomerase. Using single molecule approaches, I will assess the oligomeric state of telomerase, the biophysical properties that control its intrinsic processivity, and the impact of the interaction of TPP1 with telomerase on its catalytic cycle. The K99 phase of the proposed aims will be conducted under the mentorship of Dr. Tom Cech, who has an extraordinary track record in training post-doctoral fellows, with over 30 former mentees in faculty positions at prestigious research institutions worldwide. The Cech lab is an established leader in the biochemical and structural analysis of telomerase. In combination with my strong expertise in cell biological and microscopy-based approaches, the Cech lab provides an ideal environment to carry out the majority of the proposed research. For the proteomic analysis of shelterin assembly I will collaborate with the lab of Dr. Natalie Ahn, a leading researcher in using mass spectrometry to study protein post-translational modifications. Dr. Ahn's expertise and the proteomics core facility at the BioFrontiers Institute will allow me develop a strong foundation in using mass-spectrometry as a core discovery tool, a critical learning experience that will facilitate my short term goals and my future independent career. My goal for the K99 phase is to initiate Aims 1 and 2 of the proposal and build a strong foundation for the transition to becoming an independent investigator at a US research institution. My long term goal is to run a research program focused on the molecular mechanisms that ensure chromosomal integrity, a process defective in a large number of human diseases, using multi- disciplinary approaches including cell biological, biochemical, biophysical, proteomic, and genetic methods. A K99 grant would greatly aid me by providing critical training, helping me secure a faculty position, and allowing me to jumpstart my career as independent researcher.